Our goal is to elucidate how the architecture of the secretory pathway reflects the actions of specific molecules. This project focuses on the transitional ER (tER) and the Golgi apparatus. tER sites are membrane domains that produce COPII transport vesicles. In many eukaryotes, Golgi stacks are adjacent to tER sites. This arrangement can be understood in light of the cisternal maturation model, which postulates that tER sites give rise to new Golgi cisternae that subsequently mature. Yet little is known about how tER sites are generated, how Golgi cisternae are organized into stacks, or how Golgi stacks are positioned next to tER sites. We hypothesize that the structure of early secretory compartments is established by self-organization and influenced by the dynamics of membrane traffic. These ideas will be explored with the aid of the budding yeasts Pichia pastoris and Saccharomyces cerevisiae. P. pastoris contains large, stable tER sites that are adjacent to Golgi stacks, whereas S. cerevisiae contains fragmented tER and Golgi elements. Specific Aim #1: To test whether Sec16 defines tER sites, or regulates tER dynamics, or both. Our work has implicated the peripheral membrane protein Sec16 as a key determinant of tER organization. However, the function of Sec16 is unknown. Analyzing Sec16 will add a new dimension to our knowledge of both COPII vesicle biogenesis and tER organization. We have identified interactions between Sec16 and five COPII components. To clarify the Sec16 reaction cycle, we will use biochemical and in vivo approaches to dissect the COPII-Sec16 interactions, and to determine how these interactions regulate COPII vesicle formation and tER dynamics. An essential N-terminal region of Sec16 is important for tER localization. We will examine this localization mechanism, which may be crucial for defining tER sites. Specific Aim #2: To identify key components of the tER-Golgi matrix in budding yeasts. In most eukaryotes, a ribosome-excluding "matrix" links Golgi cisternae into stacks. In organisms such as P. pastoris, this matrix also links tER sites to the cis-Golgi, and we have now seen a similar juxtaposition of tER sites with the cis-Golgi in S. cerevisiae. We plan to identify the core components of the tER-Golgi matrix. One approach will involve microscopy- based screens in P. pastoris. A second approach will involve testing candidate matrix proteins in S. cerevisiae to identify those responsible for linking the tER to the cis-Golgi. PUBLIC HEALTH RELEVANCE: Abnormal secretory pathway function is a causative agent in diseases such as cancer and developmental disorders. Adequate treatments will require a cell biological understanding of the processes that define secretory compartments. The proposed study aims to reveal these basic principles of cellular organization.